Biochemical and histological effects of methanolic extract

IJCBS, 19(2021): 19-30

Kinjir et al., 2021 19

Biochemical and histological effects of methanolic extract of Carica

papaya seeds in Albino Wister rats infected with Trypanosomal brucei

brucei

H. Kinjir*, S. Sarkiyayi and M.A. Madusolumuo

*1Department of Biochemistry, Modibbo Adama University of Technology, Yola, Adamawa, Nigeria

Abstract

In this study, the biochemical and Histological effect of methanolic extract of Carica papaya seed in Albino Wister Rats were

determined. Eighty Wister rats of both sexes, randomly divided into 8 groups, were used. Exactly 100 mg/kg, 200mg/kg,

400mg/kg and 800mg/kg of the plant extract was administered to each group for the experimental rats infected with Trypanosomal

brucei brucei for period of 5 days. At the end of the experimental period, animals were sacrificed and blood collected by cardiac

puncture. Biochemical parameters were determined. The level of serum biochemical parameter shows significantly P<0.05

increased in AST at the concentration of 100mg/kg 200mg/kg, 400mg/kg, 800mg/kg, berenil drug control and negative control

when compared with normal control and extract control. While significantly increased in serum level of ALT and ALP were

observed at concentration of 400mg/kg, 800mg/kg, berenil drug control and negative control when compared with normal control

and extract control. The elevation in serum biochemical parameter in this study could be as a result of tissue inflammation c aused

by the parasite infection. Histopathological examination of selected organs (liver and brain) revealed histological alterations

among different treated groups with their respective controls. The histology of the brain Sections from Suprachiasmatic region of

brain. All treatment groups show mild diffuse infiltration by chronic infiltrates and increased kupfer cell population. The negative

control shows proliferation glial cells and fewer neuronal cell bodies exhibiting shrunken (pyknotic) nuclei.

Keywords: Biochemical parameters, Histological Changes, Extract and methanolic

Full length article *Corresponding Author, e-mail: [email protected] and [email protected]

1. Introduction

The use of herbal medicines in human and animal

health care systems is well documented in ancient literature.

In many parts of the World, ethno-therapies are no longer

seen as myth, superstition, witchcraft or ungodly practices

and indeed is gaining popularity with the belief that “natural

is better”. It is believed that nearly 80% of world population

relies primarily on herbal remedies for the treatment of

human and animal diseases. It is in this light that World

Health Organization encourages the use of herbal

preparations for the treatment of some local health problems

particularly in developing countries where they are readily

available, easily affordable and already integrated into the

people’s cultures. Several medicinal plants have been

proved beneficial through extensive laboratory tests [1].

Carica papaya is regarded as a wholesome fruit, the daily

requirements of some of the essential nutrients like proteins,

minerals and vitamins can be met from this fruit. The

vitamin C content increases as the maturity progresses. Its

carbohydrate content is mainly in invert sugar which is a

form of predigested food [2]. Its main medicinal use is as a

digestive agent; it is prescribed for people who have

difficulty digesting protein and is used to break up blood

clots after surgery, which is due to the presence of enzyme

papain in the plant's latex. The latex from the trunk of the

tree is also applied externally to speed the healing of

wounds, ulcers, boils, and warts. The seed is used to expel

worm and the flower may be taken in an infusion to induce

menstruation [2]. It has been documented that black seeds of

papaya are highly beneficial in the treatment of cirrhosis of

the liver caused by alcoholism, malnutrition etc. It has also

been reported that annonaceous acetogenins derived from

the extracts of the twigs of the pawpaw tree may be good

chemotherapeutic agents for cancer as these compounds

inhibit enzymes necessary for metabolic processes in tumor

cells [2]. Aqueous extract of unripe Carica papaya has been

documented to possess antisickling properties. Oduola et al.

[2] confirmed this property and established the minimum

concentration of the unripe Carica papaya that achieved

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IJCBS, 19(2021): 19-30


maximum antisickling to be 1g/ml in physiological saline.

Solvent partitioning revealed that the antisickling agent

resides in the ethyl acetate fraction of the extract. The results

of the acute oral toxicity study in Wister albino rats showed

the LD50 of the aqueous extract of the unripe Carica papaya

to be 2520mg/kg. This plant has therapeutic uses such as

anti-parasitic, anti-amoebic, anti-microbial, anti-fertility

activity, anti-ulcerogenic, anti-fungal, antitumor,

hypolipidaemic and employ in wound-healing activity, free

radical scavenging activity, diuretic activity, uterotonic

activity [3].

This paper seeks is designed to assess the effect of

the carica papaya seeds extract on biochemical parameters

and histological changes of the liver and brain tissues of

some Wister albino rats.

2. Materials and methods

2.1. Collection of plant materials

Fresh mature seed of Carica papaya was collected

in Vom, Jos south local government area of Plateau state.

Nigeria. The plant specimen was identified and

authenticated in the herbarium unit at Federal College of

Forestry, Jos, Nigeria.

2.2. Methods

2.2.1. Preparation of plants materials

Seed of Carica papaya, was washed thoroughly

with tap water in order to remove the dust and soil particles.

Then the plant was air dried under the shade to prevent

ultra-violet rays from inactivating the chemical constituents

and was pulverized (ground into powder form) separately,

using pestle and mortar [2].

2.2.2. Methanolic extraction of seed of Carica papaya

The pulverized seed of Carica papaya, (600g) was

extracted with methanol. The maceration process was

performed at room temperature by adding 600g of the

powder form to 2600 mL of methanol it was then extracted

by cold maceration with daily shaking for three days and

was filtered using Whatman filter No.1 Paper. The filtrated

was air dried. It was harvested and weight, the dried extracts

was preserved in a desiccator [4].

2.2.3. Experimental design

Albino rats weighing between 80-120 grams of

either sex were used for the study. The albino rat was kept in

clean wire meshed cages under standard animal condition in

accordance with the recommendations in Guide for the Care

and Use of Laboratory Animal [5]. Then, the animals were

given standard feed diet and water and bilirubin during the

entire period of the experiment.

2.3. Test organism

Trypasonoma brucei (Federe strain) used for this

work was obtained from Nigeria Institute for

Trypanosomiasis Research (NITR), Vom Plateau State,

Nigeria. The parasite was maintained in their laboratory by

continuous passage in albino rats, until commencement of

the work

2.3.1. Serum, liver and brain preparation

On the last day of the extract administration, the

animals were anaesthetized and sacrificed, 5 ml of the blood

will also collected in a plane container which was used for

biochemical parameters. The blood samples were separated

using refrigerated centrifuge at 3000rmp for 5 minutes. The

plasma was harvested and stored at 4°C period to time of

used. Liver and brain tissues were removed from the rats

and trimmed down to a size of 3mm × 3mm thick and fixed

in buffered formalin solution ready for histological studies.

The tissue slides were prepared in the Laboratory.

2.3.2. Effect of extract on biochemical parameters

Serum was harvested from blood kept for 2 h at

room temperature (25-26 °C) in glass test-tubes and

centrifuged at 3000 g for 5 minutes. Albumin, total protein,

conjugated bilirubin, total bilirubin, alkaline phosphatase,

aspartate aminotransferase and alanine aminotransferase.

2.3.3. Determination of alkaline phosphatase

The principle stated that alkaline phosphatase

(ALP): The substrate p-nitrophenyl phosphate is hydrolyzed

by alkaline phosphatase from the sample in the presence of

magnesium ions, to form nitrophenol which is yellow and

can be read at 405 nm. The intensity of color produced is

proportional to the activity of alkaline phosphatase. ALP

Para nitrophenol phosphate HOH [5].

In a cuvette, 10 μl of sample was mixed with 50μl

of the reagent. The initial absorbance was read at 405nm,

and subsequently over 3minutes . The mean absorbance per

minute was used in the calculation: ALP activity (IU/l) =

2742×ΔA 405 nm/min; Where: 2742 = Extinction

coefficient; ΔA 405 nm/min = change in absorbance per

minute for the sample.

Assay of alanine transaminase (ALT) activity: The

method described by IFCC] using Randox kits. To 50μl of

the sample, 500 μl of the ALT reagent was added and mixed

in a test tube, and the initial absorbance at 340 nm was read

after 1 minute. The timer will start simultaneously and

further readings of the absorbance were taken after 1, 2, and

3 minutes. ALT activity (nm/min) = 1746 × ΔA 340nm/min,

ΔA 340 nm/min = change in absorbance per minute for the

homogenate sample, 1746 = Extinction coefficient.

Assay of aspartate transaminase (AST) activity:

The same assay method described for ALT was used with

the exception that the ALT reagent was replaced with the

AST reagent. AST activity (nm/min) = 1746 × ΔA

340nm/min; ΔA 340 nm/min = change in absorbance per

minute for the homogenate sample; 1746 = extinction co-

efficient.

IJCBS, 19(2021): 19-30


2.3.4. Determination of bilirubin

Principle of Reaction Bilirubin stated that sulfanilic acid

reacts with sodium nitrite to form diazotized sulfanilic acid.

Total bilirubin reacted with diazotized sulfanilic acid in the

present of TAB form azobilirubin [4]. 1000µl of direct

bilirubin reagent was added to a test yubr label sample blank

and tested respectively. 20µl activator direct was added into

the test only and followed by 50 µl of the serum into the

sample blank and tested. It was then mixed and incubated

for 5 minutes at room temperature. The absorbance of test

against respective blank was measured at 546nm.

Calculation with factor:

Total bilirubin: 1000µl of direct bilirubin reagent was added

to a test tube label sample blank and test respectively. 20µl

activator total was added into the test only and followed by

50 µl of the serum/calibrator into the sample blank and test.

It was then mixed and incubated for 5minutes at room

temperature. The absorbance of test against respective blank

was measured at 546nm.

Calculation with factor:

2.3.5. Determination of total protein

Principle of reactions: Cupric ions in an alkaline

medium interact with protein peptide bonds resulting in the

formation of a coloured complex [4]. 1000μl of the reagent

was added to 20μl of the sample and incubated at room

temperature for 5 minutes. The absorbance against blank

was recorded at 546nm.

2.3.6. Determination of albumin

Principle of the reaction: The measurement of

serum albumin is based on the indicator 3,3’,5,5’-

tetrabromo-m cresol sulphonephthalein (bromocresol green,

BCG) which absorbs at 578nm, the absorbance being

directly proportional to the concentration of albumin in the

sample [4]. 1000μl of the reagent was added to 10μl of the

sample and incubated at room temperature for 1 minute. The

absorbance was measure at 600nm against the blank.

2.3.7. Statistical Analysis

Values was expressed as mean ± SEM n=5.Data

was subjected to one-way analysis of variance (ANOVA),

followed by SPSS version 23. Tukey’s comparison post-hoc

test used to compare the differences between the

experimental groups. (GraphPad Software, San Diego, CA,

USA). Values of p < 0.05 was considered significant.

3. Results and discussion

3.1. Biochemical parameters of rat infected with T. brucei

and treated with Carica papaya seed methanol extract

The result of serum biochemical parameter shows

significantly P<0.05 increased in AST at the concentration

of 100mg/kg 200mg/kg, 400mg/kg, 800mg/kg, berenil drug

control and negative control when compared with normal

control and extract control. While significantly increased in

serum level of ALT and ALP were observed at

concentration of 400mg/kg, 800mg/kg, berenil drug control

and negative control when compared with normal control

and extract control. The elevation in serum biochemical

parameter in this study could be as a result of tissue

inflammation caused by the parasite infection. These results

differ from Bakari, et al., [6], which didn’t show any

significant different in AST, ALT and ALP in both the

infected and uninfected cattle but they are broadly consistent

with earlier results of Ibrahim, et al., [7] in which they

recorded increased in ALT, AST and ALP. And stated that

the changing is as a result of the trypanosomes induced

hepatocellular damage, monitor by AST, ALT and ALP

hepatic cell linkage. Although Significant P<0.05 decreased

in serum level of AST, ALT and ALP where observed in

infected and treated with different concentration of extract

when compared with negative control. These could be all

attribute to ability of the extract to reduce the level of

parasitemia and presence of antioxidant which serves as

anti-inflammatory. Not much significant difference was

observed between the extract control and the normal control.

The serum albumin and total protein concentration of

negative control decreased significantly (p < 0.05) when

compared to normal control rats. Whereas there was no

significant (p< 0.05) difference in that of the normal control,

extract control, standard drug control (Berenil) and

Prophylactic. The decreased in mean value of total serum

protein level of rats agrees with previous findings of (Allam,

et al., [8]; Katunguka-Rwakishaya et al., [9], but contradicts

observations made in sheep infected with T. brucei by

Taiwo et al., [10], who observed no change in levels of total

plasma proteins from pre-infected values at the initial stage

of the infection, but in the later stage the levels increased

significantly above pre-infection levels. The decreased could

be due to reduced protein synthesis arising from damaged

liver or as a result of excessive protein breakdown arising

from reduced feed intake as observed in all the infected

animals and also could be arise from low level of albumin

caused by catabolism, uptake of albumin by trypanosomes

or heamodilution [9]. Significant (p< 0.05) increased in

serum level of bilirubin was also observed in negative

control when compared with all the others groups. Increased

in serum level of bilirubin in this study corroborates with the

earlier findings of Ekanem and Yusuf [11] (2010). Who

IJCBS, 19(2021): 19-30


stated that Bilirubin is transported to the liver bound to

albumin, High plasma conjugated bilirubin concentration

indicates impaired hepatic excretory function.

3.2. Histopathological changes on the liver and brain of

Trypanosoma brucei brucei infected rats treated with

methanolic extract of seed of Carica papaya

The tissues of rat infected with T. brucei treated

with various concentration of extract were also subjected to

histopathological analysis for 7 to 21 days post-infection.

The section of liver tissue of negative control i.e. (infected

rat and not treated) shows a moderately preserved hepatic

lobular architecture with hepatocytes generally exhibiting

reactive nuclei. Mild diffuse chronic inflammatory infiltrates

and increased kupfer cell population are observable. There

are also extensive areas of hepatocyte necrosis, with

hepatocytes losing nuclear details these were compared with

liver of normal control and extract control rat which shows a

well-preserved hepatic lobular architecture with normal-

appearing hepatocytes. All treatment groups show mild

diffuse infiltration by chronic infiltrates and increased

kupfer cell population. The mild damaged to the liver cells

could be probably as a result of the extract causing

inhibitory effect on the trypanosomes. These observation

matches with those finding observed in earlier study by

Vincent et al., [12] who showed inhibitory activity of C.

papaya leave extract on T. evansi, the liver cell of the

infected rat returned to normal.

The histopathological investigation of the brain

Sections from Suprachiasmatic region of brain. The negative

control shows proliferation glial cells and fewer neuronal

cell bodies exhibiting shrunken (pyknotic) nuclei. Some

large reactive astrocytes surrounded the inflamed blood

vessel are also seen. A few amount of T. brucei were also

seen. This study confirmed the work of Shuaibu et al., [13]

who demonstrated the presence of T. brucei in brain

parenchyma cells of infected rat. Investigation by many

researchers had shown that the trypanosomes were able to

cross the brain blood barrier (BBB). As the disease progress,

the parasite destroyed the BBB and advances to brain [14,

15]. Others studies also indicate that the laminin

composition of the basement membrane plays an important

role in trypanosome transmigration into the CNS [16, 17].

All the treated groups did not show much liver cells damage

when compared to the normal control group, this could be as

a result of the extract that have anti-inflammatory effect.

Table 1. The Biochemical parameters of Rat infected with T. brucei and treated with Carica papaya seed methanol extract

TREATMENT

GROUPS

AST (U/L) ALT (U/L). ALP (U/L) ALBUMIN (g/dl)

100 mg/Kg 105.00±42.97* 109.00±85.42 127.17±133.74* 40.50±8.36*

200 mg/Kg 99.83±101.37* 101.50±84.77 144.50±64.27 35.50±3.39*

400 mg/Kg 108.83±39.17* 123.17±70.00* 145.00±62.71 29.00±6.23*

800 mg/kg 73.77±51.44* 102.67±58.36* 115.00±149.98 33.72±1.27*

Berenil 88.50±73.50* 92.67±58.20* 72.83±78.61 51.33±7.12*

cNormal Cont. 48.00±34.16* 25.67±3.20* 65.50±12.08* 48.00±5.90*

Neg. Control 192.67±50.54* 271.50±102.88 270.05±173.07 16.00±4.60

Extract Contr 18.50±41.74* 16.17±42.94* 46.17±27.15* 41.00±9.23*

Prophylactic 154.67±55.03 88.50±161.71 114.83±74.07* 36.83±8.68*

*Significantly different from negative control down the group at P<0.05

Values are Mean ± SEM for determinations n=7

IJCBS, 19(2021): 19-30


Table 2. The Biochemical parameters of Rat infected with T. brucei and treated with Carica papaya seed methanol extract

TREATMENT GROUPS TB (mg/dl) DB (mg/dl) TP (g/dl)

100 mg/Kg 13.00±3.95* 1.30±0.11* 44.33±10.09*

200 mg/Kg 12.17±1.17 1.22±0.08* 43.67±8.98*

400 mg/Kg 12.50±2.08* 1.25±0.11* 36.83±8.70*

800 mg/kg 13.17±1.17* 1.22±0.15* 41.83±10.17*

Berenil 21.67±3.50* 2.20±0.48* 70.50±3.94*

cNormal Cont. 14.50±5.47* 1.13±0.52* 72.50±5.32*

Neg. Control 59.33±12.44 2.65±0.75 19.50±8.62

Extract Contr 21.83±8.38* 0.55±0.45 73.17±4.36*

Prophylactic 18.00±8.46 1.32±0.21 48.17±14.93*

*Significantly different from negative control down the group at P<0.05

Values are Mean ± SEM for determinations n=7

Section of liver tissue shows a moderately preserved hepatic lobular architecture. Mild diffuse chronic inflammatory infiltra tes

(yellow arrows) and increased kupfer cell population (black arrows) are observable. There are also extensive areas of hepatoc yte

necrosis (blue arrows), with hepatocytes losing nuclear details. H&E stain.

Figure 1. Histopathology of the liver of rat infected with T. brucei and treated with 100mg/kg of Carica papaya seed extract

IJCBS, 19(2021): 19-30


Section of liver tissue shows a well preserved hepatic lobular architecture with hepatocytes exhibiting reactive nuclei (blue

arrows). There is mild dilatation and congestion of hepatic sinusoids (yellow arrows), as well as a mild diffuse infiltration by

chronic inflammatory cells (black arrows). H&E stain.


Section of liver tissue shows a moderately preserved hepatic lobular architecture with hepatocytes in zone 2 exhibiting ballo oning

degeneration (blue arrows). There is marked dilatation of hepatic sinusoids (yellow arrows), as well as a mild diffuse infilt ration

by chronic inflammatory cells (black arrows). H&E stain.


Section of liver tissue shows a poorly preserved hepatic lobular architecture with hepatocytes generally exhibiting reactive nuclei

(blue arrows). There is congestion of hepatic sinusoids (yellow arrows), as well as an increase in kupfer cell population (b lack

arrows). H&E stain.


IJCBS, 19(2021): 19-30


Section of liver tissue shows a poorly preserved hepatic lobular architecture with hepatocytes generally exhibiting balloonin g

degeneration (yellow arrows). There is also an increase in kupfer cell population . H&E stain.

Figure 5. Histopathology of the liver of rat infected with T. brucei and treated with standard drug berenil (positive control)

Section of liver tissue shows a well preserved normal-appearing hepatic lobular architecture. A few karyopyknotic hepatocytes

(yellow arrows) are also seen. H&E stain.

Figure 6. Histopathology of the liver of rat not infected with T. brucei and treated with the Carica papaya seed methanol extract

Section of liver tissue shows a moderately preserved hepatic lobular architecture with hepatocytes generally exhibit ing reactive

nuclei (blue arrows) while some hepatocytes are seen with pyknotic nuclei (yellow arrows). There is mild dilatation of hepatic

sinusoids (black arrows), as well as an increase in kupfer cell population. H&E stain

Figure 7. Histopathology of the liver of rat infected with T. brucei and not treated (negative control)

G5

100µm

IJCBS, 19(2021): 19-30


Section of liver tissue shows a well-preserved hepatic lobular architecture with normal-appearing hepatocytes A scant population

of lymphocytes (yellow arrows) are also seen. H&E stain

Figure 8. Histopathology of the liver of rat not infected with T. brucei but treated with 800mg/kg of Carica papaya seed extract.

(extract control)

Section of liver tissue shows a moderately preserved hepatic lobular architecture with hepatocytes in zone 2 exhibiting ballo oning

degeneration (blue arrows). There is marked dilatation of hepatic sinusoids (yellow arrows), as well as a mild diffuse infilt ration

by chronic inflammatory cells (black arrows). H&E stain

Figure 9. Histopathology of the liver of rat treated with 800mg/kg of Carica papaya methanol extract before infected with T.

brucei (prophylactic control)

Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation of neuronal cell

bodies was seen exhibiting shrunken (psychotic) nuclei (yellow arrows). H&E stains

Figure 10. Histopathology of the brain of rat infected with T. brucei and treated with 100mg/kg of Carica papaya seed extract

G7

100µm

100µm

Section of liver tissue shows a moderately preserved hepatic lobular architecture with hepatocytes generally exhibiting reactive nuclei (blue arrows) while some

hepatocytes are seen with pyknotic nuclei (yellow arrows). There is mild dilatation of hepatic sinusoids (black arrows), as well as an increase in kupfer cell population. H&E stain

G5

100µm

IJCBS, 19(2021): 19-30


Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation glial cells an d

neuronal cell bodies are seen exhibiting shrunken (pyknotic) nuclei (yellow arrows).


Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A few neuronal cell bodies are

seen in the upper right corner with normal nuclear features (yellow arrows). No obvious proliferation of glial cells was obse rved.


Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation glial cells an d

neuronal cell bodies are seen exhibiting shrunken (pyknotic) nuclei (yellow arrows).


IJCBS, 19(2021): 19-30


Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation glial cells and

fewer neuronal cell bodies are seen exhibiting shrunken (pyknotic) nuclei.

Figure 14. Histopathology of the brain of rat infected with T. brucei and treated with standard drug berenil (positive control)


seen with normal nuclear features. No obvious proliferation of glial cells was obs erved.

Figure 15. Histopathology of the brain of rat not infected with T. brucei and also not treated with Carica papaya seed methanol

extract. (normal control)

Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation glial cells and

fewer neuronal cell bodies are seen exhibiting shrunken (pyknotic) nuclei.

Figure 16. Histopathology of the brain of rat infected with T. brucei and not treated (negative control)

G5

100µm

120µm

IJCBS, 19(2021): 19-30



seen with normal nuclear features (yellow arrows). No obvious proliferation of glial cells was observed.

Figure 17. Histopathology of the brain of rat not infected with T. brucei but treated with 800mg/kg of Carica papaya seed extract.

(extract control)

Sections from Suprachiasmatic region of brain, showing well preserved CNS tissue architecture. A proliferation of neuronal cell

bodies was seen exhibiting shrunken (pyknotic) nuclei.

Figure 18. Show Histopathology of the brain of rat treated with Carica papaya seed methanol extract before infected with T.

brucei (Extract control)

4. Conclusions

The Parasite (Trypanosomal brucei brucei) have induced

adverse alterations in biochemical parameters such as serum

AST, ALT, ALP, Albumin, TB, DB and TP and

histopathological lesions have been observed in liver or

brain tissues of experimental animals. However, all the

treated groups did not show much liver cells damage when

compared to the normal control group, this could be as a

result of the parasite that have anti-inflammatory effect.

Acknowledgement

Authors acknowledged and appreciated the

technical support of Binta Iliyasu, Esther Ogbole, O.

Domtur Larfa Larry Obaloto O.B, Oladipo O.O, and all the

staff of Nigerian Institute for Trypanosomiasis Research.

Muhammad F. Yahaya of American University Yola, and

Jibrin Muhammad Yelwa of National Research Institute for

Chemical Technology, Zaria were also acknowledged and

appreciated.

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